Figure 4

The effect of the non-complementary 5'-tail melting temperature on TSP genotyping efficiency. Genomic DNA of known zygosity (homozygous wildtype or homozygous mutant) was used to test the genotyping efficiency of NLS primers with a core melting temperature below 50°C and a non-complementary tail that increased the overall melting temperature from 5°C to 20°C above the core melting temperature once incorporated into PCR product. In this example, an NLS primer pair with a core melting temperature of 45°C and a non-complementary tail that increased the melting temperature to 60°C once incorporated in PCR product is shown for a TSP assay configured for allele-specific PCR (LS forward 5'-GCGTCGCAAAGACAAGCTGA and reverse 5'-CCGCAGGCGAACCTTTACAT with NLS forward 5'-CCGGG ATATGTTTGGGTATCATT and reverse 5'-CCCG AACTCATGGACGCAGT). Underlined sequence indicates the non-complementary 5'-tail region. The presence of the 273 bp alternate allele genotyping product in lanes for the AA homozygotes (lanes 3, 4, 5, 12, 13 and 16) indicate the presence of the A allele. However, the presence of the 153 bp allele-specific product in these DNA samples erroneously indicates that the T allele is also present. These results suggest the 5'-tail melting temperature is too high, thereby permitting the NLS primer to engage in the first phase of amplification. M represents a pUC19/HpaII DNA size ladder.